The rabies virus (RV) is a member of the family Rhabdoviridae. Like most members of this family, RV is a non-segmented, negative stranded RNA virus whose genome codes for five viral proteins: RNA-dependent RNA polymerase (L); a nucleoprotein (N); a phosphorylated protein (P); a matrix protein (M) located on the inner side of the viral protein envelope; and an external surface glycoprotein (G). Dietzschold B et al. (1991), Crit. Rev. Immunol. 10: 427-439.
Rabies is transmitted through broken skin by the bite or scratch of an animal infected with RV. Upon exposure, RV penetrates unmyelinated peripheral nerve endings, and travels to the nerve cell body by retrograde axonal transport. RV replicates exclusively in the neurons, and finally arrives in the CNS where it causes cellular dysfunction and death of the infected animal. Rupprecht C E et al. (1987), Lab. Invest. 57: 603. As RV spreads directly from cell to cell, it can substantially evade immune recognition. Clark H F et al. (1985), in Comparative Pathology of Viral Disease, Vol. 2, (Olson R G et al., eds.), CRC Press, Boca Raton, Fla. pg. 65.
The major immune effector against rabies is the production of virus neutralizing antibodies (VNA) elicited by RV G protein. The capacity to trigger the production of VNA depends largely on the integrity of RV G protein “spikes” on the encapsulating viral envelope, which are comprised of trimers of RV G. Dietzschold B et al. (1982), J. Virol. 44: 595-602. However, nonhumoral factors such as RV antigen-specific CD4+ T-helper cells, CD8+ cytotoxic T-cells (Cox J H et al. (1977), Infect. Immun. 16: 754-759) and innate defense mechanisms (Hooper D C et al. (1998), J. Virol. 72: 3711-3719) also play an important role in the anti-rabies immune response. As these cellular and innate immune defense mechanisms are triggered by the “internal” L, N and P proteins, optimal protective immunity against rabies is conferred by live RV vaccine.
Antiviral immunity may also be related to factors affecting the pathogenesis of RV. For example, the pathogenicity of a particular RV variant is inversely correlated with RV G expression levels, and increased G protein accumulation is positively correlated with the induction of apoptosis. Galelli A et al. (2000), J. Neuro Virol. 6: 359-372; Morimoto K et al. (1999), J. Virol. 73:510-517. Also, a stronger immune response is generated with non-pathogenic RV strains as compared to pathogenic RV strains. Wiktor T J (1977), P.NA.S. USA 74: 334-338.
Enhanced apoptosis is also known to contribute to the induction of antiviral immune responses to intracellular proteins. For example, the apoptotic death of cells after viral infection can trigger powerful innate and adaptive immune responses against the infecting virus. Restifo N P (2000), Curr. Opin. Immunol. 12: 597-603. Cell injury can also release endogenous adjuvants that stimulate cytotoxic T-cell responses. Shi Y et al. (2000), P.N.A.S. USA 97:14590-14595. Moreover, apoptotic cells can trigger the maturation and antigen-presenting function of dendritic cells, and apoptotic cells are believed to release factors that induce activation of MHC class I- and Il-restricted T cells by mature dendritic cells. Chattergoon M A et al. (2000), Nat. Biotech. 18: 974-979; Rovere P et al. (1998), J. Immunol. 161: 4467-4471. Apoptotic bodies also have the ability to deliver antigens to professional antigen-presenting cells. Sasaki S et al. (2001), Nat. Biotech. 19: 543-547.
The relationship between RV G protein expression, apoptosis and enhanced RV immunity is not clear. The quantity of RV G protein expressed on the cell surface appears to be important for triggering apoptotic pathways. Substantial sequence differences between the G proteins of highly pathogenic RV's, as compared to that of attenuated, pro-apoptotic RV's, suggests that G protein qualitative factors are also important in inducing apoptosis of infected cells.
Effective recombinant live RV vaccines have been produced by replacing the non-neurotropic G protein gene in antigenically conserved, laboratory RV strains with non-pathogenic neurotropic G genes from wild-type “street virus” strains. Such attenuated “street virus” RV vaccines retain the distinct neurotropisms of the wild-type strains, and also show higher replication efficiency and G protein expression levels than the wild-type RV. However, the “street virus” RV vaccines differ greatly in their ability to induce protective immunity. Morimoto J et al. (2000), J. Neuro Virol. 6: 373-381 and WO 01/70932.
Another live attenuated RV vaccine has been produced, in which the RV psi gene is replaced with a non-viral, pro-apoptotic gene such as cytochrome c. This vaccine causes increased apoptosis of infected primary cultured mouse neurons, and mice exposed to this vaccine show an approximately three-fold increase in VNA titers as compared to those vaccinated with control RV vaccine lacking a functional cytochrome c gene. However, these recombinant vaccines rely on expression of the non-viral pro-apoptotic gene, rather than an increase in the production of RV G protein, to enhance anti-viral immunity. See WO 01/70932, supra.
Thus, there is a need for a live, non-pathogenic RV vaccine which imparts enhanced rabies immunity to a subject without the requirement for species-specific neurotropism. Desirably, enhanced immunity should derive from increased apoptosis of infected cells due to an overexpression of RV G protein, and not from the expression of non-viral, pro-apoptotic genes.